The ventricles of the heart must be multilayered to ensure sufficient pumping to sustain life. During development, multilayering commences when the wall of the primitive heart tube transitions from a smooth, monolayered myocardium into a meshwork of luminal projections called trabeculae. In zebrafish, trabeculation is first observed when cardiomyocytes (CMs) in the ventricular wall delaminate towards the lumen to seed the trabecular layer. Initiation of trabecular morphogenesis occurs in a mosaic pattern and the most recent report suggests that mechanical tension dictates fate just hours prior to delamination (at 60 hours post fertilisation [hpf]).
Here, we describe a genetic marker of cardiac trabecular identity that challenges this model and shows that trabecular fate is genetically hardwired almost a day before trabecular delamination commences. The transgenic line (Tg(cbfa2t3:gal4;uas:gfp)) exquisitely labels prospective, fated, CMs from as early as 40 hpf. The transgene is expressed in the single-layer compact myocardium and, by 6 days post fertilisation, all trabecular cardiomyocytes express the transgene, leaving transgene-negative cardiomyocytes in the compact wall. Lineage tracing through Dendra2 photoconversion or Brainbow labelling show the majority (>90%) of transgene-positive cardiomyocytes end up in the trabecular layer, confirming the transgene is a marker of trabecular fate. Furthermore, knock-in to the endogenous locus confirms cbfa2t3 is responsible for this expression. Using genetic and chemical perturbations, we find that known regulators of trabeculation do not initiate or prevent cbfa2t3 expression, suggesting the mechanisms of trabecular fate induction are distinct from trabecular morphogenesis. Finally, through loss-of-function analysis and mosaic overexpression, we show that cbfa2t3 is both necessary and sufficient to drive trabeculation and can do so in a cell-autonomous manner.
This work introduces cbfa2t3 as the earliest known marker and driver of trabeculation. Altogether, these data describe a previously unappreciated step in cardiac trabeculation; the determination of fate. Trabecular fate is temporally and genetically separate from trabecular morphogenesis.